Summary/Abstract The long term goal of the proposed studies is to understand the signal transduction mechanisms in cancer development and progression. The previous funding period focused on the role of focal adhesion kinase (FAK) and several other proteins in the regulation of cell migration and breast cancer development and metastasis. We have illustrated the mechanisms by which FAK-mediated Src phosphorylation of endophilin A2 inhibits endocytosis of MT1-MMP to stimulate cancer cell invasion. Using a conditional KO approach, we showed that mammary epithelial-specific deletion of FAK leads to severe lobulo-alveolar hypoplasia and secretory immaturity of the murine mammary gland. Furthermore, we demonstrated that inactivation of FAK in mammary epithelial cells suppresses mammary tumorigenesis and progression by affecting mammary cancer stem/progenitor cells using the well-characterized MMTV-PyMT mouse model of human breast cancer. We also created the defined fibronectin gradients using microfluidics approaches and analyzed the role of FAK and a number of other signaling molecules in the regulation of directional cell migration. Additional studies identified a novel interaction between N-WASP and hnRNPK and showed that hnRNPK may function as a negative regulator of N-WASP to inhibit filopodia formation and cell spreading. This grant also supported our work on the role of nuclear localized actin regulatory proteins N-WASP and Arp2/3 complex in the regulation of RNA polymerase II- dependent transcription. In preliminary studies, we made the unexpected finding that inactivation of FIP200, a putative breast tumor suppressor based on initial studies in cancer cell lines, inhibits (rather than promotes) mammary tumorigenesis and progression in the MMTV-PyMT mouse model of breast cancer in vivo. We further showed that both intrinsic proliferative defects of FIP200-null mammary tumor cells as well as increased infiltration of immune cells in the tumor microenvironment may contribute to the suppression of mammary tumorigenesis and progression in MMTV- PyMT mice with conditional KO of FIP200. Based on these preliminary studies, we propose to determine the mechanisms by which FIP200 and its associated signaling pathways regulate breast cancer development and progression using a combination of molecular, cellular, immunological and mouse genetic approaches. We will 1). analyze molecular mechanisms of FIP200 regulation of mammary tumor cell proliferation using primary culture of isolated tumor cells, 2). examine the role and mechanisms of elevated immune cell infiltration in the suppression of mammary tumor development upon FIP200 ablation, and 3). investigate the potential role of FIP200 in mammary tumor maintenance.